The incidence of breast cancer in the United States in 2010 was 209,060, and the number of deaths was 40,230 (National Cancer Institute U.S. National Institutes of Health). Estrogen receptor negative breast cancer refers to a specific subtype of breast cancer that does not express the genes for estrogen or progesterone receptors. Triple-negative breast cancer refers to a specific subtype that does not express the genes for estrogen receptor, progesterone receptor or HER-2/ERBb2 (Her2/neu) receptor. About 85% of primary tumors are estrogen receptor positive; however about 71% of circulating tumor cells are estrogen receptor negative and 50% of circulating tumor cells are triple negative. The expression of Muc4, a high molecular weight glycoprotein on mammary epithelial cells, is upregulated with aberrant expression in over 95% of breast tumors: 40% in solid tumors and 70% in malignant effusions. These tumor-associated Muc4 expressing cells have aggressive clinical pathologic features associated with poor prognosis and elevated risk of recurrence. The risk of distant recurrence in women with triple-negative breast cancer is between two and three times that found in women with other breast cancers such as estrogen-receptor positive and HER-2/ERBb2 (Her2/neu) receptor positive (Dent R. et al. Clinical Cancer Research 13(15):4429-4434 (2007)). The most successful breast cancer treatments use drugs that directly target estrogen receptors. Because estrogen-negative breast cancer cells do not express these receptors, they are unresponsive to these treatments. Few, if any, effective clinical prospects exist for modulating the uncontrolled expression of Muc4 in over 95% of breast tumors and the aggressive clinical pathology of Muc4-containing cells.
The Muc4 glycoprotein is the human homologue of rat sialomucin complex (SMC). Sialomucin complex was originally isolated from highly malignant metastatic 13762 rat mammary adenocarcinoma ascites cells. It is a heterodimeric tumor cell surface glycoprotein composed of a high molecular weight peripheral sialomucin (ascites sialoglycoprotein-1, ASGP-1) tightly but noncovalently linked to a 120 kDa transmembrane N-glycosylated component ASGP-2. ASGP-1 is the major detectable glycoprotein that extends outwardly from the cell surface; whereas ASGP-2 is a slightly asymmetric globular protein having a Stokes radius of 4.6 nm. ASGP-2 contains two EGF-like domains and has been shown to act as a ligand for the tyrosine kinase p185neu. Sialomucin complex is present at very high levels in the ascites cells (>106 copies/cell), and the sialomucin has been implicated in the resistance to natural killer (NK) cells that play a major role in the rejection of tumors.
Muc4 consists of two subunits, the extracellular highly glycosylated mucin subunit (MUC4-α) with an extended rigid extracellular domain that can extend up to 2 micrometer over the cell membrane and a transmembrane subunit (Muc-4-β) containing three EGF-like domains. MUC4 is translated as a single precursor polypeptide, which is further cleaved at a GDPH site in two subunits before addition of O-linked oligosaccharides (Komatsu M. et al. Biochemical Journal 368:41-48 (2002)). Most normal tissues expressing Muc4/SMC produce both membrane bound and soluble forms of Muc4; however in malignant transformed rat tumors, Muc4/SMC is expressed predominantly in a membrane-bound form. It has been reported that ascites 13762 rat mammary adenocarcinoma cell surface sialomucin ASGP-1 is synthesized initially as a poorly glycosylated immature form, which is converted to a larger premature form with a t1/2 of about 30 minutes and more slowly to the mature glycoprotein through O-glycosylation with a t1/2 greater than 4 hours. Although 95% of ASGP-1 reaches the cell surface in 2 h, ASGP-1 reaches the cell surface in an incompletely glycosylated state, and additional oligosaccharides are added to the glycoprotein after ASGP-1 has reached the cell surface in a second process involving recycling and resialylation (Hull S. et al Journal of Biological Chemistry 25; 266(21):13580-13586 (1991)).
Muc4 is poorly expressed on the apical surface of mammary epithelial cells; however, it is overexpressed on a number of human breast tumors. Muc4 is expressed in a minority of solid breast tumors and is overexpressed in the majority of more aggressive tumor cells from malignant effusions. Overexpression of Muc4 has been shown to block cell-cell and cell-matrix interactions, protect tumor cells from immune surveillance and promote metastasis. Muc4 is also the putative ligand of the HER-2/ERBb2 (Her2/neu) oncogenic receptor found in about 25% of invasive breast cancers. Muc4 binds to and modulates phosphorylation of the HER-2/ERBb2 (Her2/neu) receptor and influences cell proliferation, tumor progression, and tumor cell morphology; however, overexpression of Muc4 does not interfere with surface expression of ErbB2 receptors. Overexpression of Muc4 sterically blocks anti-ErbB2 antibody binding to HER-2/ERBb2 (Her2/neu) oncogenic receptors such as seen with Herceptin (trastuzumab), possibly reducing its therapeutic effect. The expression of Muc4 protein is inversely correlated with trastuzumab binding capacity, it masks the binding epitopes of HER2 antibody, and it masks the epitope interaction of the HER-2/ERBb2 (Her2/neu) oncogenic receptor with other proteins such as EGRF (International Journal of Cancer 99:783-791 (2002)).
The glycan remodeling in breast cancer cells is complex when Muc4 is overexpressed. Compared with O-glycans from normal mucins whose O-glycans have an extended GlcNacβ1-6(Galβ1-3)GalNacα-Ser/Thr (core-2) structure, breast cancer-associated O-glycans are highly sialylated and sialylated Galβ1-3(Galβ1-3)GalNacα-Ser/Thr (core-1) prevails. An increase in the expression of α3-sialyltransferase ST3Gal-1, which acts on Galβ1-3(Galβ1-3)GalNacα-Ser/Thr (core 1), is characteristic of breast cancer cells that are most likely to metastasize (Brockhausen I EMBP Reports 7(6): 599-604 (2006)). Antibodies to the negatively charged sialylated epitopes or desialylation of mucin glycoproteins on the outer surface of the transformed cell have been shown to inhibit adhesion of metastatic cells to basement membranes.
The expression of Muc4 in stably transfected human ovarian cancer cells was analyzed by western blot, confocal microscopy and in vitro motility of transformed cells (Ponnusamy M P et al. British Journal of Cancer 99: 520-526 (2008). These Muc4-expressing cells demonstrated a significantly enhanced motility compared to control cells (P<0.05) and exhibited significant morphological changes associated with coordinated disassembly and reformation of the cortical actin organization. The increased motility is consistent with previous observations that silencing of Muc4 expression led to a three-fold decrease in cell motility (Singh, A P et al. Cancer Research 64, 622-630, Jan. 15, 2004). Muc4 plays an important role in tumor cell growth, behavior, and metastasis. Silencing Muc4 expression in cells that expresses high levels of MUC4 results in 1) a 3-fold decrease in the observed in vitro cell motility compared with control cells; 2) a significant decrease in tumor growth and metastatic properties when transplanted orthotopically into immunodeficient mice; and 3) reduced expression of its putative interacting partner, HER-2/ERBb2 (Her2/neu) (Bafna, S. et al. Cancer Research 68(22):9231-8 (2008)). Moreover, the in vivo tumorigenicity of Muc4-transfected cells injected in nude mice is significantly greater than with Muc4-deficient control cells. The mean tumor-free survival time over 40 days was significantly less (P<0.001) with injected Muc4-transfected cells (17.6 days) than with Muc4-deficient control cells (34.7 days). Only 7% of the mice injected with Muc4-transfected cells showed tumor-free survival at the end of 36 days, whereas 73% of the mice injected with Muc4-deficient control cells showed tumor-free survival at the end of 36 days. Tumor volume was consistently and significantly less with injected Muc4-deficient control cells than with injection of Muc4-transfected cells.
Published studies have demonstrated that the epithelial to mesenchymal transition of tumor-associated Muc4 is inhibited by TGF-β ALK5 inhibitors. These studies show that the TGF-β-induced epithelial to mesenchymal transition (EMT) in normal (NMuMG) cells was mediated though activation of the ALK5 receptor and Smad2 effector proteins. Inhibition of the phosphorylation of Smad2 by TGF-β ALK5 inhibitors was associated with less cell motility and cell morphological changes associated with EMT and inhibition of TGF-β signaling reduced Muc4 protein expression. These studies are consistent with other studies showing that silencing Muc4 expression results in a significant decrease in cell motility and metastatic properties in vivo. Studies have shown that the epithelial to mesenchymal transition in the normal mammary epithelial cells stimulated by TGF-β was abrogated by a general serine/threonine kinase and protein kinase inhibitor, staurosporine (Piek, E et al. Journal of Cell Science 112:4557-4568 (1999)), and that the TGF-β-induced epithelial to mesenchymal transition of NMuMG cells was mediated though activation of the ALK5 receptor and Smad2 effector proteins. TGF-β strongly induces Smad2 phosphorylation and its nuclear translocation. Using a series of specific small molecule inhibitors, it has been shown that only the general serine/threonine kinase and protein kinase inhibitor staurosporine exhibited a major effect on cell morphology and the expression of E-cadherin and β-catenin which render cells more motile and invasive. This finding was replicated in the regulation of the transformed sialomucin complex/rat Muc4 in mammary epithelial cells. Muc4 protein expression in transfected mammary epithelial cells (MEC) in a serum-free medium is low compared to Muc4 protein expression in 10% fetal calf serum, and Muc4 protein expression is inhibited by staurosporine (protein kinase C inhibitor-operating through TGF-β and Smad2) when activated in fetal calf serum (Zhu X. et al. Oncogene 19: 4354-4361 (2000)). Finally, it has been reported that only TGF-β1, TGF-β2, TGF-β3 among all type I TGF-β type receptors (TGF-β1, TGF-β2, TGF-β3, activin-A or BMP-7) induce EMT and growth inhibition in normal human cells (MCF-10A) and normal mouse epithelial cells (NMuMG) through the ALK5 mediated Smad pathway involving Smad2 (Valcourt U. et al. Molecular Biology of the Cell 16:1987-2002 (2002)). The selective ALK5 inhibitor SB431542 ((4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), a potent and selective inhibitor of the transforming growth factor-β (TGF-β) type I receptor activin receptor-like kinase ALK5 having an IC50=94 nM, inhibited the phosphorylation of Smad2 and morphological changes associated with EMT in NMuMG cells stimulated with TGF-β1.
A major disadvantage of nonselective small molecule inhibitors is their inherent cross-reactivity with other kinases. Given the exceptionally broad range of biological activities associated with TGF-β and its fundamental physiological roles, it is reasonable that nonselective TGF-β blockade could lead to loss of immune tolerance with uncontrolled activation of T and B cells. One benefit of using specific Smad2 regulated ALK5 inhibitors is the ability to select Smad-specific affecting the epithelial to mesenchymal transition responses in tumor cells without affecting other TGF-β signaling pathways; however, ALK5/Smad regulated TGF-β signaling is not limited to regulation in tumor cells (Goumans et al. Trends Cardiovascular Medicine 13(7):301-307 (2003); Lebrin F et al. Cardiovascular Research 65(3):599-608 (2005); Tsuchida K et al. Endocr. J. 55(1):11-21 (2008); Gauldie J et al. Biochem Soc Trans 35(Pt 4):661-4 (2007)). Because Muc4-expressing tumor cells do not express any specific protein receptors, selectively targeting these tumor cells and delivering specific inhibitors to regulate the Smad2 pathway is currently not possible.
It is an object of the present invention to provide compounds and methods for treating a subject having cancer comprising administering to the subject a tumor-targeted TGF-β ALK5 inhibitor-glycoconjugate in an amount effective in selectively targeting tumor-associated Muc4 glycoproteins, delivering TGF-β ALK5 inhibitors to the tumor and regulating the expression levels and glycosylation of tumor-associated Muc4.
It is a further object of the present invention to provide compounds and methods for modulating the epithelial-mesenchymal-transition of tumor cells and the progression of the cancer by reducing the migration, extravasation and metastatic dissemination of mesenchymal tumor cells.
It is a further object of the present invention to provide compounds and methods for treating a subject having a cancer in need of therapy thereof by administering to the subject a Muc4-targeted compound in an amount effective in reducing Muc4 expression or glycosylation.